Altered excitatory and inhibitory neocortical circuitry leads to increased convulsive severity after pentylenetetrazol injection in an animal model of schizencephaly, but not of microgyria.

OBJECTIVE: Malformations of the polymicrogyria spectrum can be mimicked in rodents through neonatal transcranial focal cortical freeze lesions. The animals presenting the malformations present both altered synaptic events and epileptiform activity in the vicinity of the microgyrus, but the comprehension of their contribution to increased predisposition or severity of seizures require further studies. METHODS: In order to investigate these issues, we induced both microgyria and schizencephaly in 57 mice and evaluated: their convulsive susceptibility and severity after pentyleneterazol (PTZ) treatment, the quantification of their symmetric and asymmetric synapses, the morphology of their dendritic arbors, and the content of modulators of synaptogenesis, such as SPARC, gephyrin and GAP-43 within the adjacent visual cortex. RESULTS: Our results have shown that only schizencephalic animals present increased convulsive severity. Nevertheless, both microgyric and schizencephalic cortices present increased synapse number and dendritic complexity of layer IV and layer V-located neurons. Specifically, the microgyric cortex presented reduced inhibitory synapses, while the schizencephalic cortex presented increased excitatory synapses. This altered synapse number is correlated with decreased content of both the anti-synaptogenic factor SPARC and the inhibitory postsynaptic organizer gephyrin in both malformed groups. Besides, GAP-43 content and dendritic spines number are enhanced exclusively in schizencephalic cortices. SIGNIFICANCE: In conclusion, our study supports the hypothesis that the sum of synaptic alterations drives to convulsive aggravation in animals with schizencephaly, but not microgyria after PTZ treatment. These findings reveal that different malformations of cortical development should trigger epilepsy via different mechanisms, requiring further studies for development of specific therapeutic interventions.

Epigenetic control of myeloid cells behavior by Histone Deacetylase activity (HDAC) during tissue and organ regeneration in Xenopus laevis.

In the present work we have focused on the Histone Deacetylase (HDAC) control of myeloid cells behavior during Xenopus tail regeneration. Here we show that myeloid differentiation is crucial to modulate the regenerative ability of Xenopus tadpoles in a HDAC activity-dependent fashion. HDAC activity inhibition during the first wave of myeloid differentiation disrupted myeloid cells dynamics in the regenerative bud as well the mRNA expression pattern of myeloid markers, such as LURP, MPOX, Spib and mmp7. We also functionally bridge the spatial and temporal dynamics of lipid droplets, the main platform of lipid mediators synthesis in myeloid cells during the inflammatory response, and the regenerative ability of Xenopus tadpoles. In addition, we showed that 15-LOX activity is necessary during tail regeneration. Taken together our results support a role for the epigenetic control of myeloid behavior during tissue and organ regeneration, which may positively impact translational approaches for regenerative medicine.

Novel CCM1 (KRIT1) Mutation Detection in Brazilian Familial Cerebral Cavernous Malformation: Different Genetic Variants in Inflammation, Oxidative Stress, and Drug Metabolism Genes Affect Disease Aggressiveness.

BACKGROUND: Cerebral cavernous malformations (CCMs) are vascular capillary anomalies with a dysfunctional endothelial adherent junction profile, depicting hemorrhage and epilepsy as the main clinical features. With the advent of an increasingly personalized medicine, better comprehension of genetic mechanisms behind CCM represents an important key in the management of the patients and risk rating in relatives. In this context, genetic factors that might influence clinical expressiveness of CCM need to be identified. CASE DESCRIPTION: A 33-year-old woman harboring multiple CCM lesions with a CCM1 mutational profile already being treated conservatively for a right mesial temporal lobe CCM presented with refractory seizures. Magnetic resonance imaging showed no bleeding in the lesion, and the patient was submitted to complete resection of the CCM. Histopathology of the CCM samples depicted an extensive inflammatory reaction and colocalization of CD20+ and CD68+ cells. Genetic analyses of the patient and her mother demonstrated a novel CCM1 (KRIT1) frameshift mutation (c.1661_1662insT; p.Leu554PhefsTer14). Furthermore, variants in CD14 (rs778588), TLR-4 (rs10759930), SOD2 (rs4880), APEX1 (rs1130409), and OGG1 (rs1052133), known as polymorphisms related to disease aggressiveness, were detected in the patient and not in her oligosymptomatic mother harboring the same CCM1 mutation. CONCLUSIONS: Heterogeneity of clinical manifestations among individuals with familial CCM with the same genotype adds mechanistic involvement of modifier factors as phenotypic markers. We describe a novel CCM1/KRIT1 familial mutation in which the coexistence of genetic variants in inflammation and oxidative stress may be related to variable expressiveness of the disease.

Neuroinflammation and oxidative stress act in concert to promote neurodegeneration in the diabetic retina and optic nerve: galectin-3 participation.

Diabetes is a lifelong disease characterized by glucose metabolic imbalance, in which low insulin levels or impaired insulin signaling lead to hyperglycemic state. Within 20 years of diabetes progression, 95% of patients will have diabetic retinopathy, the leading cause of visual defects in working-age people worldwide. Although diabetes is considered a microvascular disease, recent studies have shown that neurodegeneration precedes vascular changes within the diabetic visual system, albeit its mechanisms are still under investigation. Neuroinflammation and oxidative stress are intrinsically related phenomena, since macrophage/microglia and astrocytes are the main sources of reactive oxygen species during central nervous system chronic degenerative diseases, and both pathological processes are increased in the visual system during diabetes. The present review will focus on recent findings of the contribution of oxidative stress derived from neuroinflammation in the early neurodegenerative aspects of the diabetic visual system and their relationship with galectin-3.

Inosine Accelerates the Regeneration and Anticipates the Functional Recovery after Sciatic Nerve Crush Injury in Mice.

Trauma to the peripheral nervous system (PNS) results in loss of motor and sensory functions. After an injury, a complex series of events begins, allowing axonal regeneration and target reinnervation. However, this regenerative potential is limited by several factors such as age, distance from the lesion site to the target and severity of lesion. Many studies look for ways to overcome these limitations. Inosine, a purine nucleoside derived from adenosine, emerges as a potential treatment, due to its capacity to regulate axonal growth, neuroprotection and immunomodulation, contributing to motor recovery. However, no studies demonstrated their effects on PNS. C57/Black6 mice were submitted to sciatic nerve crush and received intraperitoneal injections of saline or inosine (70 mg/kg), one hour after injury and daily for one week. To evaluate axonal regeneration and functional recovery, electroneuromyography, Sciatic Function Index (SFI), rotarod and pinprick tests were performed. Our results showed that the inosine group presented a higher number of myelinated fibers and a large amount of fibers within the ideal G-ratio. In addition, the results of electroneuromyography showed greater amplitude of the compound muscle action potentials in the first and second weeks, suggesting anticipation of regeneration in the inosine group. We also observed in the inosine group, motor and sensory neurons survival, reduction in the number of macrophages and myelin ovoids in the sciatic nerves, and an early recovery of motor and sensory functions. Thus, we conclude that the use of inosine accelerates axonal regeneration promoting an early recovery of motor and sensory functions.

Intraaxial and Extraaxial Cavernous Malformation with Venous Linkage: Immune Cellular Inflammation Associated with Aggressiveness.

BACKGROUND: Intraorbital and intracerebral cavernous malformation (CM) lesions are considered independent entities. Purely cerebral CMs have variable biology with recent evidence depicting inflammation as an important player and a risk factor for aggressiveness. We describe a case of concomitant left intraaxial and extraaxial CMs, linked by the ipsilateral basal vein, where the extraaxial component has developed an aggressive behavior. CASE DESCRIPTION: A 35-year-old female patient presented with a rapid and progressive exophthalmos and loss of vision on the left eye. Cranial magnetic resonance and angiography examinations demonstrated a left craniofacial CM and large intraorbital component. The lesion was connected through a large basal vein to a cerebral intraventricular CM. Transconjunctival resection showed typical findings of CM. A complete histopathology and immunostaining analysis was performed and revealed a clear acute lymphomononuclear reaction with a predominant immune cellular inflammation. CONCLUSIONS: A case of intraorbital and extracranial cavernomatous mass, connected to a cerebral intraventricular CM through a large basal vein, has presented with an aggressive course. A complete histopathologic and immunohistochemical analysis of the orbital mass has pictured a clear immune-cellular inflammatory reaction adding to the amounting evidence of association between inflammation and site aggressiveness in the setting of CMs.

Effects of Different Doses of Mesenchymal Stem Cells on Functional Recovery After Compressive Spinal-Cord Injury in Mice.

Despite advances in technology and rehabilitation, no effective therapies are available for patients with SCI, which remains a major medical challenge. This study compared the efficacy of 3 different doses of mesenchymal stem cells (MSCs) administered by intraperitoneal injection as a therapeutic strategy for compressive SCI. We used adult female C57BL/6 mice that underwent laminectomy at the T9 level, followed by spinal-cord compression for 1 min with a 30-g vascular clip. The animals received an intraperitoneal (i.p.) injection of MSCs (8 × 104, 8 × 105 or 8 × 106 in 500 µl) or DMEM (500 µl), one week after SCI. The cells of the three MSC doses administered i.p. were able to migrate to the injury site, increase local expression of trophic factors, and enhance fiber sparing and/or regeneration, accompanied by substantial improvement in locomotor performance. Cell transplantation at 8 × 105 density showed the best therapeutic potential, leading to significant tissue and functional improvements compared to the other two doses. These findings indicate that i.p. application of MSCs at the density of 8 × 105 yielded the best results, suggesting that this dose is a good choice for SCI treatment.

Olfactory ensheathing glia cell therapy and tubular conduit enhance nerve regeneration after mouse sciatic nerve transection.

The regenerative potential of the peripheral nervous system (PNS) is widely known, but functional recovery, particularly in humans, is seldom complete. Therefore, it is necessary to resort to strategies that induce or potentiate the PNS regeneration. Our main objective was to test the effectiveness of Olfactory Ensheathing Cells (OEC) transplantation into a biodegradable conduit as a therapeutic strategy to improve the repair outcome after nerve injury. Sciatic nerve transection was performed in C57BL/6 mice; proximal and distal stumps of the nerve were sutured into the collagen conduit. Two groups were analyzed: DMEM (acellular grafts) and OEC (1×105/2µL). Locomotor function was assessed weekly by Sciatic Function Index (SFI) and Global Mobility Test (GMT). After eight weeks the sciatic nerve was dissected for morphological analysis. Our results showed that the OEC group exhibited many clusters of regenerated nerve fibers, a higher number of myelinated fibers and myelin area compared to DMEM group. The G-ratio analysis of the OEC group showed significantly more fibers on the most suitable sciatic nerve G-ratio index. Motor recovery was accelerated in the OEC group. These data provide evidence that the OEC therapy can improve sciatic nerve functional and morphological recovery and can be potentially translated to the clinical setting.

Bone marrow stromal cells and resorbable collagen guidance tubes enhance sciatic nerve regeneration in mice.

We evaluated peripheral nerve regeneration using a tubular nerve guide of resorbable collagen filled with either bone marrow-derived cells (BMDCs) in Dulbecco's cell culture medium (DMEM) or with DMEM alone (control). The control group received just the culture medium (vehicle). The left sciatic nerves of ten isogenic mice were transected and the tubular nerve guides were sutured to the end of the proximal and distal nerve stumps. Motor function was tested at 2, 4 and 6 weeks after surgery using the walking track test. The pawprints were analyzed and the print lengths (PL) were measured to evaluate functional recovery. After 6 weeks, mice were anesthetized, perfused transcardially with fixative containing aldehydes, and the sciatic nerves and tubes were dissected and processed for scanning and transmission electron microscopy. Scanning electron microscopy of the collagen tube revealed that the tube wall became progressively thinner after surgery, proving that the tube can be resorbed in vivo. Quantitative analysis of the regenerating nerves showed that the number of myelinated fibers and the myelin area were significantly increased in the experimental group. Also, motor function recovery was faster in animals that received the cell grafts. These results indicate that the collagen tube filled with BMDCs provided an adequate and favorable environment for the growth and myelination of regenerating axons compared to the collagen tube alone.

Calpain inhibitor 2 prevents axonal degeneration of opossum optic nerve fibers.

The ultrastructural change that characterizes the onset of Wallerian degeneration is the disintegration of axoplasmic microtubules and neurofilaments, which are converted into an amorphous and granular material, followed by myelin breakdown. The mechanism underlying such processes is an increase in the amount of intracellular calcium, leading to activation of proteases called calpains. The aim of this study was to evaluate by quantitative ultrastructural analysis whether nerve fibers can be preserved by the use of an exogenous inhibitor of these proteases (calpain inhibitor-2, Mu-F-hF-FMK), after optic nerve crush. For that, the left optic nerves of opossums, Didelphis aurita, were crushed with the aid of a fine forceps, and half of them received a calpain inhibitor mixed with Elvax resin. Ninety-six hours after the lesion, the animals were reanesthetized and transcardially perfused, and the optic nerves were removed, the right ones being used as normal nerves. Afterward, the optic nerves were dissected and processed for routine transmission electron microscopy and quantitative and statistical analysis. The results of this analysis showed that the group that received the calpain inhibitor presented a reduction of astrogliosis, maintaining the optic nerve structure in an organized state; a significant decrease in the number of degenerating fibers; and a significant increase in the number of fibers with preserved cytoskeleton and preservation of axonal and myelin area and integrity, reducing the enlargement and edema of the axon. In conclusion, our findings suggest that calpain inhibitor is able to provide neuroprotection of the central nervous system fibers after a crush lesion.

Participation of neurofilament proteins in axonal dark degeneration of rat's optic nerves.

Neurofilaments (NF) are neuronal intermediate filaments formed by three different subunits: high (NF-H), medium (NF-M) and light (NF-L). They are responsible for the determination and maintenance of axon caliber. Accumulation of NF or their immunoreactive products are components of several neurodegenerative disease lesions, such as neurofibrillary tangles, Lewy bodies and the spheroids of amyotrophic lateral sclerosis. Also, cytoskeletal breakdown is one of the first ultrastructural changes occurring after nerve crush or section. In the present study, Wistar rats were subjected to bilateral enucleation to induce Wallerian degeneration of optic nerve fibers and perfused 24 h, 48 h and 1 week later. Optic nerve segments were processed for electron microscopy (EM), light microscopy immunofluorescence (LM) and immunoelectronmicroscopy (IEM) for NF subunit detection. LM for NF of control nerves showed a slightly different pattern and intensity for each subunit, with more intense staining of NF-M and NF-H and less intense staining of NF-L. This reaction did not change considerably at 48 h, but was severely reduced 1 week after enucleation. Results of EM showed fibers in: (1) partial cytoskeleton degeneration or (2) watery degeneration or (3) dark degeneration. The number of dark degenerating axons was statistically higher at the latest time-interval studied. Neurofilament clumping areas and dark degenerating axons showed positive immunostaining for the three neurofilaments subunits when examined by IEM. These results suggest that dark degenerating axons develop from areas of neurofilament aggregation. We may also conclude that NF proteins participate in the process of axonal dark degeneration.